G proteins ? subunits play a central role in G-protein coupled receptor (GPCR)-mediated signal transduction. They act as cofactors in the receptor-mediated activation process as well as playing direct roles in signal transfer to downstream targets. Considerable data has accumulated in number of systems that excess ? signaling has pathological consequences and that manipulation of ? subunit signaling could be an effective therapeutic strategy in heart failure as well as other diseases. We developed a novel targeting strategy for selective manipulation of G protein ? subunit signaling pathways by selectively blocking ? -subunit binding interactions with functional protein partners using small molecules. In the previous funding period we defined the binding modes for several compounds by surface plasmon resonance (SPR) coupled with site directed mutagenesis and solved the co-crystal structure of M201 bound to the hot spot of G?. These data confirmed a direct mechanism for binding to G? that influences protein-protein interactions and support our overall hypothesis that small molecules selectively modulate downstream effectors signaling by binding to different subsites on the G? hotspot. Additionally, we published results demonstrating efficacy and specificity of these compounds in cellular and animal models of heart failure, inflammation and morphine- dependent analgesia. In the experiments proposed in this application we will continue to explore the fundamental mechanisms underlying binding and selectivity of these ? binding compounds. Specific aim 1 will focus on mutagenesis and x-ray crystallography to identify multiple binding modes within the G? hotspot that contribute to selectivity. Specific aim 2 will explore the mechanism for compound-dependent G? subunit activation. Specific aim 3 will explore specificity and mechanism of action in intact cells. Successful completion of the proposed experiments will lead to a thorough understanding of a the mechanism of action of a new family of molecules that target G23 signaling that have potential uses in dissecting the mechanisms of action of GPCR stimulated signaling and providing the basis for novel therapeutic approaches. PUBLIC HEALTH RELEVANCE: G protein coupled receptors (GPCRs) are a major class of transmembrane receptors responsible for recognition of a large class of diverse ligands. Here we propose investigation of selective small molecule inhibitors of G protein ? subunits identified in our laboratory which could be used to inhibit multiple GPCRs and modify actions of existing GPCR directed pharmaceuticals. Results of these experiments will help to validate this alternate approach to modification of signaling pathways downstream of GPCRs that could ultimately lead to development of novel therapeutics.